Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Patent
1994-08-17
1998-04-07
Wax, Robert A.
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
435212, 4353201, 435325, 4352523, 536 232, A61K 3848, C12N 948, C12N 121, C07H 2104
Patent
active
057361347
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention is directed to particular novel variants of tissue plasminogen activator (t-PA). These variants, although embraced generically by earlier disclosure, as noted infra, are novel, specific derivatives which exhibit activities which defied prediction from the prior research of others on the basic tissue plasminogen activator molecule or the model serine proteases trypsin and chymotrypsin.
As the preferred embodiment herein contemplates the preparation of such variants via recombinant DNA technology, the present invention likewise encompasses the associated compounds and means involved in such technology, namely, DNA isolates encoding such variants, expression vectors, transfected host cells and processes for making and using each of them. The novel variants hereof have unexpectedly enhanced fibrin specificity.
BACKGROUND OF THE INVENTION
Plasminogen activators are enzymes that activate the zymogen plasminogen to generate the serine proteinase plasmin (by cleavage at Arg561-Va1562) that degrades various proteins, including fibrin. Among the plasminogen activators studied are streptokinase, a bacterial protein, urokinase, an enzyme synthesized in the kidney and elsewhere and originally extracted from urine, and tissue plasminogen activator (t-PA), an enzyme produced by the cells lining blood vessel walls.
The mechanism of action of each of these plasminogen activators differs: Streptokinase forms a complex with plasminogen or plasmin, generating plasminogen-activating activity, urokinase cleaves plasminogen directly, and t-PA requires fibrin to yield maximum plasminogen activating activity.
t-PA has been identified and described as a particularly important and potent new biological pharmaceutical agent that has shown extraordinary results in the treatment of vascular diseases, such as myocardial infarction and pulmonary embolism, due in part to its fibrin specificity and potent ability to dissolve blood clots in vivo.
Tissue plasminogen activator was first identified as a substantially pure isolate from a natural source, and tested for requisite plasminogen activator activity by Collen, et al., European Patent Application Publication No. 041766, published 16 Dec. 1981, based upon a first filing of 11 Jun. 1980. See also U.S. Pat. No. 4,752,603, issued 21 Jun. 1988 and Rijken, et al., J. Biol. Chem. 256, 7035 (1981).
Subsequently, researchers in Assignee's laboratories produced large quantities of tissue plasminogen activator, fully characterized by underlying DNA and deduced amino acid sequences, essentially free of proteins with which it is ordinarily associated, in a distinct milieu, via recombinant DNA technology. This work has been recorded in the scientific literature and in European Patent Application Publication No. 093619 published 9 Nov. 1983, based upon a first filing on 5 May 1982. See also U.S. Pat. No. 4,766,075, issued 23 Aug. 1988 and Pennica, et al., Nature 301, 214 (1983). The above patent application (EPO Publication No. 093619) contemplates the production of various human plasminogen activator derivatives, variously modified by amino acid substitutions, deletions, additions, or replacements prepared, for example, by site directed mutagenesis of the underlying DNA.
For further patent literature regarding modification of the protease cleavage site of t-PA, see, for example, EPO Pat. Nos. 241,209; EP 201,153 published Nov. 12, 1986; EP 233,013 published Aug. 19, 1987; EP 293,936 published Dec. 7, 1988; and EP 293,934 published Dec. 7, 1988; and WO 88/10119.
EP 292009, published 23 Nov. 1988 (priority 22 May 1987) refers to t-PA "homologs" including t-PAs having certain amino acid substitutions at positions 274-277. Other more radical homologs are described. A single 276 "homolog" has proline substituted for isoleucine.
It would be desirable to provide a t-PA molecule that, relative to wild-type t-PA, has a higher ratio of fibrin-stimulated (or a plasma clot-stimulated) activity to fibrinogen-stimulated (or plasma-stimulated) activity, i.e., is more
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Bennet, W., et al., "High Resolution Analysis of Functional Determinants on Human Tissue-type Plasminogen Activator," J. Biol. Chem. 266:5191 (1991).
Pennica, D., et al., "Cloning and Expression of Human Tissue-type Plasminogen Activator cDNA in E. coli," Nature 301:214 (1983).
Tate, K., et al., "Functional Role of Proteolytic Cleavage in Arginine-275 of Human Tissue Plasminogen Activator as Assessed by Site-directed Mutagenesis," Biochemistry 26:338 (1987).
Bennett William F.
Heyneker Herbert L.
Paoni Nicholas F.
Vehar Gordon A.
Dreger Walter H.
Genentech, In.c
Lau Kawai
Wax Robert A.
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